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Low phosphate media forBacillus/Bacillus transformation protocol - (Mar/27/2001 )

Hi, I`m looking for a protocol which yields a good amount of the very-low-copy cosmid DNA. I`ve been trying every possible way I could do, but somehow those were not successful. The size of Cosmid itself is around 14-kb, and its insert is 27-kb which makes 41-kb altogether.(It`s a bacterial library clone.)I need about 100 microgram to work, but with the protocols I`ve tried,no way. I did CsCl-gradient prep, scale-up mini-prep with repeated Phenol:Chloroform extract to remove proteins and RNase, and Qiagen midi-prep. The midi was quite Ok, but we don`t have any more column, and we can`t afford it right at this moment. Besides, even if we order the column, we need to wait another 1-2 weeks to recieve it in Korea. The scale-up prep was not bad either, but it was not clean enough to work. ( I`m also wondering how on earth there was so much RNA survived... The DNA sample in TE was treated with enough amount of RNase though...!!)

So, simply the question is that,

"Is there any good way developed in your own lab without using commercial kits?" ....AND WHICH CAN YIELD A GOOD AMOUNT OF *CLEAN* DNA.

Now I`m preparing the sample in 2-liter of superbroth, 1-liter will be proceeded as scale-up mini prep except treating RNase twice with sol'n I and after dissolving the DNA in TE, and the other using PEG.

If you have experienced in this problem, please, please~~~! show me the way...... :) I`m so desperate..

Good luck on your research! *^o^*

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If you have the used Qiagen-column it is possible to use it twice - but only for the same DNA.
Ive tried it several times and it worked fine.

What is your next aim?
For restriction analysis and cloning there is enough cosmid-DNA yielded in Mdid-Prrp without using a column.

Greetings
Wotan

-Wotan-